PROJECT SUMMARY Infection with Human Immunodeficiency virus (HIV)-1 is frequently associated with abuse of psychostimulant drugs, such as methamphetamine (METH). The interaction of virus and psychostimulant, in particular with regard to viral persistence, is poorly understood and will be studied here using in vitro and in vivo approaches. We recently observed that blockade of p38 MAPK and knockdown or deletion of the lncRNA linc02574-201 can inhibit HIV-1 replication. Therefore, we propose in this application to investigate the mechanism(s) by which METH apparently promotes HIV-1 infection and the potential contributions of p38 MAPK and lncRNA linc02574- 201. Three Specific Aims are proposed: 1) To investigate how methamphetamine (METH) increases HIV-1 infection of peripheral blood lymphocytes and monocytes/macrophages. This aim will test the hypothesis that METH exposure affects HIV-1 infection in a concentration- and timing-dependent fashion by promoting the activity of p38 MAPK and expression of lnRNA linc02574-201. 2) To study how METH interferes with viral suppression by combined antiretroviral therapy (cART). This aim will investigate how METH exposure affects viral suppression by cART and the recrudescence of the virus once ARV treatment ceases. This aims will also assess if inhibition or knockdown of p38 MAPK can prevent the resumption of viral replication after cART, similar to the knockdown of lncRNA linc02574-201. 3) To assess in a humanized CD34+ HSC-engrafted NSG mouse model if METH increases the viral reservoir and facilitates the recurrence of HIV during interruption of cART. This aim will study how METH affects HIV-1 infection, viral suppression by cART and viral recrudescence upon interruption of cART in an HIV permissive in vivo model, the CD34+ HSC (hematopoietic stem cell)-engrafted NSG mouse model. The experiments will be performed in vitro with isolated human peripheral blood cells and cell lines, and in vivo in humanized mice with a CD34+ cell-derived hematopoietic system that provides HIV permissive human peripheral CD4+ T-lymphocytes and macrophages (M?). All three Specific Aims will define RNA signatures of HIV-1 infected CD4+ T-cells and M? in the presence and absence of METH using RNA- sequencing and will test the proposed mechanism, including increased activity of the stress-related p38 MAPK and up-regulation of lncRNA linc02574-201. The aims will also monitor factors known to support HIV-1 infection, such as IRF7, and will test the premise that METH exposure down-regulates a subset of ISGs that include antiviral factors, such as CCL5, Mx1/2, IFITM2/3 and IRF1 and -3.